解放军医学杂志2012，Vol.37Issue(4)：300-303,4.
人野生型和突变型CITED2真核表达质粒的构建与表达
Construction and expression of human wild-type and mutant CITED2 in eukaryotic expression plasmid
摘要
Abstract
Objective To construct eukaryotic plasmids encoding wild-type or mutant (c.573-578del6) human transcription factor CITED2, and determine the expression level of CITED2 protein. Methods Coding strands of CITED2, including wild-type and mutant-type, were obtained by PCR directional cloning methods from blood samples of healthy children and children with congenital heart diseases, and then transfected into pMD19-T simple vector by T/A cloning technique, and the recombinant plasmids of mutant and wild CITED2 T vector were screened. The CITED2 gene fragments in recombinant plasmids of T vector were subcloned into eukaryotic expression vector pEGFP-Cl using DNA recombinant technique to construct eukaryotic expression plasmids of pEGFP-Cl-wtCITED2 and pEGFP-Cl-mtCITED2. Then the constructed eukaryotic expression plasmids were transfected into HEK293 cells, the expression of EGFP was observed by fluorescence microscopy 24 hours later, the transfection efficiency was determined by flow cytometry, and CITED2 protein expression determined by Western blotting 48 hours later. Results Eukaryotic expression plasmids of pEGFP-Cl-wtCITED2 (wild type) and pEGFP-Cl-mtCITED2 (mutant type) were constructed and transfected into HEK293 cells. Twenty-four hours after transfection, EGFP signal was observed by fluorescence microscopy in HEK293 cells; 48 hours after transfection, the transfection efficiency was 50%-60% as detected by flow cytometry, and the fusion expression of CITED2 and EGFP was confirmed by Western blotting. Conclusion The eukaryotic expression plasmids of wild and mutant CITED2 were successfully constructed, and the CITED2 protein expression was detected.关键词
基因,CITED2/突变/心脏缺损,先天性/真核表达质粒Key words
genes,CITED2/mutation/heart defects,congenital/eukaryotic expression vector分类
医药卫生引用本文复制引用
胡计华,吴晓云,杨晓菲,郑敏,邓兵,田杰..人野生型和突变型CITED2真核表达质粒的构建与表达[J].解放军医学杂志,2012,37(4):300-303,4.基金项目
重庆市卫生局医学科研计划(2009-2-259) （2009-2-259）
重庆市自然科学基金(2008BB5071) （2008BB5071）
国家人力资源和社会保障部留学人员科技活动择优资助启动项目[渝人社办(2009)116号] （2009）
